Dimensional Liquid Chromatography Research Articles

Gastric biomarker study using proteomic method

Biomarker is defined as biological variables that correlate with biologic outcome. This review will discuss investigations into gastric cancer (GC) biomarkers by proteomic analysis. Proteomic analysis consists of 3 steps. The first step is the digestion and separation process using 2-dimensional electrophoresis gel or liquid chromatography. The second step is mass analysis using mass spectrometry. The third step is protein identification using databases. Clinical validation of proteins identified can help estimate expressions of cancer tissue and cancer cell line using Western blot and immunohistochemistry. Researchers can validate the association between protein expression and clinical data (tumor stage, cell type, survival, and recurrence), which helps identify the possibility of biomarkers for GC. After clinical validation, the next step is functional analysis in vitro and in vivo. This step is commonly performed by knock-in and knock-out studies on the proliferation, migration, and invasion using the cancer cell line. Animal studies also provide indirect evidence for the role of the proteins in tumor growth and metastasis in vivo. In conclusion, the proteomic analysis is one of the useful methods for detecting biomarkers for GC. Multidisciplinary approaches to protein, DNA, RNA, and epigenetics are crucial to the investigation for molecular biomarkers for GC. Keywords: Stomach neoplasms, Biomarkers, Proteomics, Immunohistochemistry, Tissue array analysis

On-line comprehensive two-dimensional normal-phase liquid chromatography × reversed-phase liquid chromatography for preparative isolation of toad venom

An on-line comprehensive preparative two–dimensional normal-phase liquid chromatography×reversed-phase liquid chromatography (2D NPLC×RPLC) system was constructed with a newly developed vacuum evaporation assisted adsorption (VEAA) interface, allowing fast removal of NPLC solvent in the vacuum condition and successfully solving the solvent incompatibility problem between NPLC and RPLC. The system achieved on-line solvent exchange within the two dimensions and its performance was illustrated by gram-scale isolation of crude extract from the venom of Bufo bufo gargarizans. Within separation time of ∼20h, 19 compounds were obtained with high purity in a single run. With the VEAA interface, the 2D system exhibited apparent advantages in separation efficiency and automation compared with conventional methods, indicating its promising application in the routine separation process for complicated natural products.

Investigation of low recovery in the free drug assay for antibody drug conjugates by size exclusion-reversed phase two dimensional-liquid chromatography.

Antibody drug conjugates (ADCs) are complex therapeutic agents combining the selectivity of monoclonal antibodies and highly efficacious small molecule drugs to successfully eliminate tumor cells while limiting the general toxicity and side effects of the therapeutic to protect patient safety. One unique attribute critical to the safety of ADCs is the residual content of unconjugated small molecule drug present from either incomplete conjugation or degradation of the ADC. Typically for quality control assays, quantifying the amount of the free drug is performed through precipitation of the protein species using an organic solvent and then assaying the amount of free drug left in the supernatant. During the validation of an assay of this type for a maleimide based linker drug, issues were experienced with low and variable recovery in the spiked samples of the drug substance and drug product. A two-dimensional heart-cutting method coupling Size Exclusion Chromatography (SEC) with Reverse Phase (RP) chromatography was utilized to explore possible mechanisms leading to the low recovery of the free linker drug. The results of the investigation indicated that the spiked linker drug reacts with residual reactive groups on the ADC; a conclusion which was confirmed by the observed increase of average Drug to Antibody Ratio (DAR) determined by Hydrophobic Interaction Chromatography (HIC). Finally, several approaches were evaluated to minimize the recovery loss. Capping the residual reactive groups on the ADC with maleimide containing reagents effectively mitigated the low recovery issue.

Analysis of histone post translational modifications in primary monocyte derived macrophages using reverse phase × reverse phase chromatography in conjunction with porous graphitic carbon stationary phase

A two dimensional-liquid chromatography (2D-LC) based approach was developed for the identification and quantification of histone post translational modifications in conjunction with mass spectrometry analysis. Using a bottom-up strategy, offline 2D-LC was developed using reverse phase chromatography. A porous graphitic carbon stationary phase in the first dimension and a C18 stationary phase in the second dimension interfaced with mass spectrometry was used to analyse global levels of histone post translational modifications in human primary monocyte-derived macrophages. The results demonstrated that 84 different histone peptide proteoforms, with modifications at 18 different sites including combinatorial marks were identified, representing an increase in the identification of histone peptides by 65% and 51% compared to two different 1D-LC approaches on the same mass spectrometer.The use of the porous graphitic stationary phase in the first dimension resulted in efficient separation of histone peptides across the gradient, with good resolution and is orthogonal to the online C18 reverse phase chromatography. Overall, more histone peptides were identified using the 2D-LC approach compared to conventional 1D-LC approaches. In addition, a bioinformatic pipeline was developed in-house to enable the high throughput efficient and accurate quantification of fractionated histone peptides. The automation of a section of the downstream analysis pipeline increased the throughput of the 2D-LC–MS/MS approach for the quantification of histone post translational modifications.

Simultaneous enantioselective quantification of fluoxetine and norfluoxetine in human milk by direct sample injection using 2-dimensional liquid chromatography–tandem mass spectrometry

A two-dimensional liquid chromatography system coupled to triple quadrupole tandem mass spectrometer (2D LC–MS/MS) was employed for the simultaneously quantification of fluoxetine (FLX) and norfluoxetine (NFLX) enantiomers in human milk by direct injection of samples. A restricted access media of bovine serum albumin octadecyl column (RAM-BSAC18) was used in the first dimension for the milk proteins depletion, while an antibiotic-based chiral column was used in the second dimension. The results herein described show good selectivity, extraction efficiency, accuracy, and precision with limits of quantification in the order of 7.5ngmL−1for the FLX enantiomers and 10.0ngmL−1 for NFLX enantiomers. Furthermore, it represents a practical tool in terms of sustainability for the sample preparation of such a difficult matrix.

Phosphorylation of Neuronal Proteins in Drosophila melanogaster Changes With Age

Cells, such as epithetical and red blood cells, have the capacity to regenerate throughout an organism's lifetime. However, neurons are unique because they do not regenerate; once most neurons are formed, they last the lifetime of an organism. This lack of regeneration is the reason that neurodegenerative diseases, including Alzheimer's Disease and Huntington's Disease, are so detrimental; the diseases target cells that can't be replaced if they're damaged. These diseases occur later in life, which indicates a problem with the normal aging process of neurons; these diseases are associated with abnormal protein accumulation. There are housekeeping genes that are associated with normal cell upkeep; this upkeep can happen by protein degradation by either proteosomal digestion or autophagy. The two degradation pathways are quality control mechanisms that allow for degradation of abnormal or old proteins without destroying the undamaged proteins, which allows for longevity in structures such as neurons. As neurons age, they acquire intracellular aggregates despite these degradation pathways, and some of these aggregates are key features of neurodegenerative diseases. We are interested in understanding how well degradation pathways work in neurons of different ages, and what changes in older cells lead to intracellular aggregates. One key way to understand how neurons age is to investigate how neuronal proteins change over time. Changes in protein abundance or post‐translational modification of existing proteins could severely affect how cells deal with the stresses of aging. To determine how proteins change with age, protein levels and post‐translational modifications are compared between young and old neurons to determine how the same protein could differ over time. The changes are then analyzed in order to identify post‐translational modifications that are important for healthy neuronal aging and investigate the functional significance of the modifications. We have focused on identifying changes in protein phosphorylation between young and old brains in wild type Drosophila melanogaster by two dimensional gel electrophoresis and liquid chromatography mass spectroscopy. Preliminary results have found increased levels of protein phosphorylation in old Drosophila melanogaster heads. Once we analyze these findings through mass spectroscopy, we will be able to identify the exact proteins that differ in order to study their function in Drosophila.

Array-Based Online Two Dimensional Liquid Chromatography System Applied to Effective Depletion of High-Abundance Proteins in Human Plasma.

In this work, an array-based online two-dimensional liquid chromatography (2D-LC) system was constructed for protein separation and effective depletion of high-abundance proteins in human plasma. This system employed a strong anion exchange column in the first dimension and eight reversed-phase liquid chromatographic columns in the second dimension. All the protein components in the first dimension were enriched on the trapping columns, simultaneously back-flushed and concurrently separated in the second dimension. LC eluents were then collected on 96-well plates for further analysis. Compared with common 2D-LC system, this system showed an 8-fold increase in throughput and convenient utilization of stop-flow mode for sample separation. The RSD of retention time and peak area were separately below 0.51% and 8%. Recovery rates of four standard proteins were all above 95%. This array-based 2D-LC system was subsequently applied to the analysis of proteins in human plasma. The eluents containing high-abundance proteins were rapidly located according to the results of bicinchoninic acid assay. In all, with the effective depletion of 84 high-abundance proteins, a total of 1332 proteins were identified through our system. The dynamic range of the identified protein concentrations covered 9 orders of magnitude, ranging from 41 g/L level for HSA down to 0.01 ng/mL level for the low-abundance proteins.

Abstract B13: Discovery of novel subtype-specific ovarian cancer biomarkers via integrated tissue proteomics.

Abstract Introduction and Objectives: It is evident that ovarian cancer (OvCa) is not a single disease but is made up of several distinct subtypes, including serous, endometrioid, clear cell and mucinous. The gold-standard biomarker CA125 performs well in serous but not in the other histotypes. We hypothesize that a more focused discovery effort (on non-serous OvCa biomarkers and/or markers that can complement serum CA125) may bring about a sensitivity that is acceptable for all histotypes and be suitable for early diagnosis of OvCa. Methods: Tissues from patients diagnosed with endometrioid (EC), clear cell (CC), and mucinous ovarian carcinoma (MC), as well as from their appropriate controls (endometriosis and healthy endometrium for EC and CC; mucinous cystadenoma for MC) were subjected to proteomic analysis using a label-free, offline 2-dimensional liquid chromatography tandem mass spectrometry-based approach. Discovery candidates were then filtered using an in-house developed algorithm combining publicly-available resources with our own warehouse of transcriptomic and proteomic sets. Results and Discussion: Over 8000 unique proteins were identified in this proteomic exercise; specifically, approximately 1500 protein unique to MC, 1100 unique to CC, and 3000 unique to EC were identified when comparing the appropriate cases and controls. A curated list of 60 high-potential candidates was generated after using a range of bioinformatics tools to ensure criteria based on factors including (but not limited to) tissue specificity, cellular localization and transcriptional upregulation were met. These 60 candidates represent putative subtype-specific markers which will be further analyzed and validated in serum cohorts. Conclusions: The identification and validation of markers specific to the non-serous subtypes of OvCa remains an unmet clinical need. With our list of putative subtype-specific markers, we aim to develop a novel biomarker panel able to detect all OvCa histotypes with greater sensitivity and specificity than any existing clinical tools. Citation Format: Felix Leung, Marcus Q. Bernardini, Blaise Clarke, Marjan Rouzbahman, Eleftherios P. Diamandis, Vathany Kulasingam. Discovery of novel subtype-specific ovarian cancer biomarkers via integrated tissue proteomics. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research: Exploiting Vulnerabilities; Oct 17-20, 2015; Orlando, FL. Philadelphia (PA): AACR; Clin Cancer Res 2016;22(2 Suppl):Abstract nr B13.

Abstract B46: The use of quantitative proteomics to increase the knowledge about metabolic symbiosis in a tumor microenvironment

Abstract For more than a century, it is known that in a solid tumor, malignant cancer cells co-reside with stromal cells including fibroblasts and immune cells. In addition, changes in metabolic requirements of malignant cells are necessary to cover their high proliferative rate. Although it has been demonstrated that complementary metabolic functions of tumor cells and their supporting local stroma (e.g. cancer-associated fibroblasts, adipocytes) contribute to the progression of cancer, less is known about the metabolic needs of immune cells in this local tumor microenvironment. However, in the context of cancer, immune cells, including tumor-associated macrophages (TAMs) are of high interest since it is known that these cells are able to influence tumor progression by allowing the establishment of an attractive environment for tumor growth and metastasis. Studying the expression profiles of proteins involved in metabolic processes of these cells might be a good strategy to elucidate this question. In this study, we applied quantitative proteomics to study the differences in protein expression of TAMs vs. control macrophages (both bone marrow-derived and peritoneal macrophages) isolated from an in vivo murine model for non-small cell lung adenocarcinoma. In our peptide-centric two dimensional liquid chromatography (LC)- tandem mass spectrometry (MS/MS) setup where Tandem Mass Tag (TMT) labeling is applied, we were able to identify 1250 unique proteins, of which 1178 proteins were quantified with all six TMT reporter ions. Interestingly, analysis of the differentially expressed proteins indicate that several Gene Ontology terms regarding metabolic pathways including glutathione metabolic processes (p= 3.47 e-14) and pentose phosphate shunt (p= 2.29 e-8) were enriched. These data suggest that TAMs can reprogram their metabolism in order to survive to the oxidative stress existing in this kind of microenvironment. In conclusion, our results illustrate that proteome analysis is a good strategy to obtain insights in the biology of tumor-associated macrophages. Citation Format: Evelyne Maes, Mathias Wenes, Dirk Valkenborg, Inge Mertens, Hans Prenen, Massimiliano Mazzone, Geert Baggerman. The use of quantitative proteomics to increase the knowledge about metabolic symbiosis in a tumor microenvironment. [abstract]. In: Proceedings of the AACR Special Conference: Metabolism and Cancer; Jun 7-10, 2015; Bellevue, WA. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(1_Suppl):Abstract nr B46.

Advances and Changes in the Techniques of Multi- Dimensional and Comprehensive Chromatography and When Coupled with Mass Spectrometry

This review covers the principles of two dimensional gas and liquid chromatography (2DGC and 2DLC) and briefly introduces the theory accounting for the increase in separation resulting from a greater peak capacity than for the one dimensional (1D) mode. The advance in the techniques from multi-dimensional to comprehensive chromatography is discussed. The more recent development of multi-dimensional chromatography ion mobility mass spectrometry receives a mention to highlight the added dimension of molecular size and shape (molecular collision cross section) as an enabling tool for increasing component separation and peak capacity. Although both the techniques of 2DGC and LC are described the focus is on the environmental and food applications of 2DGC, principally when coupled to mass spectrometry, with TOFMS and HRMS as prime examples.

Unraveling the effects of static magnetic field stress on cytosolic proteins of Salmonella by using a proteomic approach.

The present study investigated the adaptation of Salmonella enterica subsp. enterica serovar Hadar to static magnetic field (SMF) exposure (200 mT, 9 h). The proteomic analysis provides an overview of potentially important cytosolic proteins that Salmonella needs to regulate to survive and adapt to magnetic stress. Via 2-dimensional electrophoresis and liquid chromatography tandem mass spectrometry, we compared cytosolic proteomes before and after exposure to magnetic field. A total of 35 proteins displaying more than a 2-fold change were differentially expressed in exposed cells, among which 25 were upregulated and 10 were downregulated. These proteins can be classified mainly into 6 categories: (i) proteins involved in metabolic pathways of carbohydrates, (ii) chaperones and proteins produced in response to oxidative stress, (iii) proteins involved in energy homeostasis, (iv) elongation factors (EF-Tu and EF-Ts), (v) proteins involved in motility, and (vi) proteins involved in molecules transport. Many of the presented observations could be explained, while some represent still-unknown mechanisms. In addition, this study reveals 5 hypothetical proteins. It seems that the stress response to SMF (200 mT) is essentially set up to avoid oxidative damages, with the overexpression of proteins directly involved in oxidative stress response and metabolic switches to counteract oxidative stress. Interestingly, several proteins induced under SMF exposure are found to overlap with those induced by other stresses, such as heat shock and starvation.

Characterization and quantification of histidine degradation in therapeutic protein formulations by size exclusion-hydrophilic interaction two dimensional-liquid chromatography with stable-isotope labeling mass spectrometry

Two dimensional liquid chromatography (2D-LC) coupling size exclusion (SEC) and hydrophilic interaction chromatography (HILIC) is demonstrated as a useful tool to study polar excipients, such as histidine and its degradant, in protein formulation samples. The SEC-HILIC setup successfully removed interferences from complex sample matrices and enabled accurate mass measurement of the histidine degradation product, which was then determined to be trans-urocanic acid. Because the SEC effluent is a strong solvent for the second dimension HILIC, experimental parameters needed to be carefully chosen, i.e., small transferring loop, fast gradient at high flow rates for the second dimension gradient, in order to mitigate the solvent mismatch and to ensure good peak shapes for HILIC separations. In addition, the generation of trans-urocanic acid was quantified by single heart-cutting SEC-HILIC 2D-LC combined with stable-isotope labeling mass spectrometry. Compared with existing 2D quantification methods, the proposed approach is fast, insensitive to solvent mismatch between dimensions, and tolerant of small retention time shifts in the first dimension. Finally, the first dimension diode array detector was found to be a potential degradation source for photolabile analytes such as trans-urocanic acid.

Array-Based Two Dimensional Liquid Chromatography System for Proteomic Analysis of Human Plasma

Abstract The human plasma proteome has the characteristics of complexity in component and large dynamic range of protein concentrations. Herein, an array-based online two dimensional liquid chromatography system combined with protein equalizer technology was developed for the large-scale depletion of high abundance proteins and enrichment of low abundance proteins in human plasma. The array-based online two dimensional liquid chromatography system could be used to separate the plasma at the intact protein level with good reproducibility and high throughput. The total separation time was only 4 h and the fast location of high abundance proteins was also achieved. After the high abundance protein fractions was treated by ampholine@PM polymer microsphere, the number of identified low abundance proteins increased ten-fold, which significantly decreased the loss of low abundance proteins in high abundance protein fractions. The techniques combined were then applied to perform the proteomic analysis of human plasma sample. The total number of identified proteins was 1474 and the dynamic range of protein concentration was 7. In this work, 252 proteins were identified in high abundance protein fractions, among which 61 proteins belonged to high abundance proteins. These results demonstrated that an array-based online two dimensional liquid chromatography system combined with protein equalizer technology could efficiently achieve the large-scale depletion of high abundance proteins and the enrichment of low abundance proteins in human plasma, with a remarkable improvement in protein identification and a great prospect in the proteomic research of other complex samples.

Assessment of global proteome in LNCaP cells by 2D-RP/RP LC–MS/MS following sulforaphane exposure

The phytochemical sulforaphane can induce cell cycle arrest and apoptosis in metastatic prostate cancer cells, though the mechanism of action is not fully known. We conducted a global proteome analysis in LNCaP metastatic prostate cancer cells to characterize how global protein signature responds to sulforaphane. We conducted parallel analyses to evaluate semi-quantitative 1-dimensional versus 2-dimensional liquid chromatography tandem mass spectrometry (LC–MS/MS) and their utility in characterizing whole cell lysate. We show that 2-dimensional LC–MS/MS can be a useful tool for characterizing global protein profiles and identify TRIAP1 as a novel regulator of cell proliferation in LNCaP metastatic prostate cancer cells.

Endoplasmic reticulium protein profiling of heat-stressed Jurkat cells by one dimensional electrophoresis and liquid chromatography tandem mass spectrometry.

Proteomic study on membrane-integrated proteins in endoplasmic reticulum (ER) fractions was performed. In this study, we examined the effects of heat stress on Jurkat cells. The ER fractions were highly purified by differential centrifugation with sodium carbonate washing and acetone methanol precipitations. The ER membrane proteins were separated by one dimensional electrophoresis (1-DE), and some of the protein bands changed their abundance by heat stress, 12 of the 14 bands containing 40 and 60 ribosomal proteins whose expression level were decreased, on the contrary, 2 of the 14 bands containing ubiquitin and eukaryotic translation initiation factor 3 were increased. Heat treatment of human Jurkat cells led to an increase in the phosphorylation of PERK and eIF2α within 30min of exposure. This was followed by an increase in the expression of the GRP78. Protein ubiquitination and subsequent degradation by the proteasome are important mechanisms regulating cell cycle, growth and differentiation, the result showed that heat stress enhanced ubiquitination modification of the microsomal proteins. The data of this study strongly suggest that heat treatment led to a significant reduction in protein expression and activated UPR, concomitant with protein hyperubiqutination in ER.

Three dimensional liquid chromatography coupling ion exchange chromatography/hydrophobic interaction chromatography/reverse phase chromatography for effective protein separation in top-down proteomics.

To address the complexity of the proteome in mass spectrometry (MS)-based top-down proteomics, multidimensional liquid chromatography (MDLC) strategies that can effectively separate proteins with high resolution and automation are highly desirable. Although various MDLC methods that can effectively separate peptides from protein digests exist, very few MDLC strategies, primarily consisting of 2DLC, are available for intact protein separation, which is insufficient to address the complexity of the proteome. We recently demonstrated that hydrophobic interaction chromatography (HIC) utilizing a MS-compatible salt can provide high resolution separation of intact proteins for top-down proteomics. Herein, we have developed a novel 3DLC strategy by coupling HIC with ion exchange chromatography (IEC) and reverse phase chromatography (RPC) for intact protein separation. We demonstrated that a 3D (IEC-HIC-RPC) approach greatly outperformed the conventional 2D IEC-RPC approach. For the same IEC fraction (out of 35 fractions) from a crude HEK 293 cell lysate, a total of 640 proteins were identified in the 3D approach (corresponding to 201 nonredundant proteins) as compared to 47 in the 2D approach, whereas simply prolonging the gradients in RPC in the 2D approach only led to minimal improvement in protein separation and identifications. Therefore, this novel 3DLC method has great potential for effective separation of intact proteins to achieve deep proteome coverage in top-down proteomics.

Two dimensional separations of human urinary protein digest using a droplet-interfaced platform

For highly complex mixtures, coelution is a common phenomenon in chromatography. A great deal of resolution is hidden in coelution, and lost due to inevitable molecular diffusion during sample transfer. The molecular diffusion may lead to band broadening and remix of separated peaks, which cause degradation of achievable resolution. In this study, we introduced droplet microfluidics as a high performance sample transfer tool in two dimensional nanoflow liquid chromatography–capillary electrophoresis separation of a human urine sample. The fine fractionation capability and sampling completeness enabled by the droplet-interface demonstrated the 2D system’s usefulness in high-resolution mapping of real world biological samples.

Double Off-line Two-dimensional Liquid Chromatography for Separation and Identification of Compounds in Salvia Miltiorrhiza (Danshen)

Background: Danshen is an important traditional Chinese medicine (TCM) used for the treatment of cardiovascular and cerebrovascular diseases. Separation and analysis of its components have been widely investigated. However, the systematical two dimensional liquid chromatography (2D-LC) methods have not been developed to comprehensively separate and characterize its components. Objective: In this work, double off-line 2D-LC methods were aimed to develop for the systematical separation of compounds from Danshen. Methods: Using solid phase extraction (SPE), the Danshen extract was divided into a medium-polar fraction (Sample I) and a weak-polar fraction (Sample II) according to their polarities. Based on reversed-phase liquid chromatography (RPLC) and hydrophilic interaction liquid chromatography (HILIC) modes, a 2D-HILIC × RPLC system and a 2D-RPLC × RPLC system were designed for the separation of Sample I and Sample II, respectively. According to reversed-phase and HILIC columns selectivities characterized in our previous reports, ZIC-HILIC and XTerra C18 were employed to build the 2D-HILIC × RPLC system and Click TE-CD and XTerra C18 for the 2D-RPLC × RPLC system, respectively. Results: The 2D-HILIC × RPLC and 2D-RPLC × RPLC systems exhibited excellent orthogonality for the separation of Sample I and Sample II, respectively. Their orthogonalities were 88.42% and 63.24%. Based on these double 2D-LC systems combined with mass spectrometry, at least 200 compounds were found and 33 compounds of them were identified, including 16 phenolic acids and 17 diterpenoid quinines. Conclusion: These results suggest that these two off-line 2D-LC methods are effective for the separation and characterization of components in Danshen.

Proteomic network analysis of human uterine smooth muscle in pregnancy, labor, and preterm labor.

The molecular mechanisms involved in human uterine quiescence during gestation and the induction of labor at term or preterm are not completely known. Preterm delivery is associated with major morbidity and mortality and current efforts to prevent delivery until term are largely ineffective. Identification and semi-quantification of proteomic changes in uterine smooth muscle during pregnancy will allow for targeted research into how quiescence is maintained and what changes are associated with induction of labor. Examining preterm labor in this context will provide potential therapeutic targets for the management of preterm labor. We have recently performed two dimensional liquid chromatography coupled with tandem mass spectrometry on myometrial proteins isolated from pregnant patients in labor, pregnant patients not in labor, and pregnant patients in labor preterm. Using a conservative false discovery rate of 1% we have identified 2132 protein groups using this method and semi-quantitative spectral counting shows 201 proteins that have disparate levels of expression in preterm laboring samples. To our knowledge this is the first large scale proteomic study examining human uterine smooth muscle and this initial work has provided a target list for future experiments that can address how changing protein levels are involved in the induction of labor at term and preterm.

Analytical Performance of LC-MS/MS Method for Simultaneous Determination of Five Steroids in Serum

The accurate measurement of steroid hormones is the fulcrum of modern endocrinology. Our aim was to develop a mass spectrometry coupled with a liquid chromatographic method for 17-hydroxyprogesterone, androstenedione, 11-desoxycortisol, 21-desoxycortisol and cortisol serum quantification simultaneously. The samples were submitted to isotopic internal standards addition, protein precipitation and two dimensional liquid chromatography consisting of trapping column and reverse-phase C18 analytical column following atmospheric pressure chemical ionization and mass spectrometry detection. For all compounds tested functional sensitivity was less than 0.5 ng/mL, precision was less than 15%, recovery ranged from 93% to 120%, linearity ranged from 89% to 111%, and accuracy was considered adequate. In conclusion, we developed a suitable, fast and reliable method for simultaneous routine measurement of 17-hydroxyprogesterone, androstenedione, 11-desoxycortisol, 21-desoxycortisol and cortisol in serum.